Electroosmotic and electrodialysis concentration,purification,and desalting

ABSTRACT

HOG CHOLERA SERUM AND ITS FRACTIONS, AND GENERALLY AQUEOUS COLLOIDAL SOLUTIONS AND SUSPENSIONS ARE CONCENTRATED, PURIFIED, AND/OR DESALTED BY SUBJECTING THEM TO ELECTROOSMOSIS AGAINST AN AQUEOUS SUSPENSION CONTAINING A POLYIONIC SUBSTANCE.

M. BIER July 1s, 1972 Y ELECTROOSMOTIC AND ELECTRODIALYSIS CONCENTRATION,

PURIFICATION, AND DESALTING 2 Sheets-Sheet 1 Filed April 15, 1.968

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l l /l/l/l/ll/l/ [lll l lll /lll July 18, 1972 M. BIER. 3,677,923

ELECTROOSMOTIC AND ELECTRODIALYSIS CONCENTRATION,

PURIFICATION, AND DESALTING Filed April l5, 1968 2 Sheets-Sheet 2 United States Patent U.S. Cl. 204-180 P 15 Claims ABSTRACT OF THE DISCLOSURE Hog cholera serum and its fractions, and generally aqueous colloidal solutions and suspensions are concentrated, purified, and/or desalted by subjecting them to electroosmosis against an aqueous suspension containing a polyionic substance.

The invention relates to a process and apparatus for the electroosmotic concentration, purification, and/or desalting of hog cholera antiserum, its fractions, and of other aqueous solutions or suspensions.

Fractions of hog cholera antiserum are obtainable by the process defined in my Pat. No. 3,079,318, but there they are obtained in a dilute state. Their concentration by said process is possible, but may result in substantial losses, While other methods, such as freeze drying, are expensive. The process herein disclosed permits the concentration of said fractions and generally of colloidal suspensions or solutions and even of salt solutions, to be carried out without losses, and in an economic manner.

A principal object of the invention is to provide an electroosmotic process which makes it possible to concentrate hog cholera antiserum, its fractions, or other colloidal materials of biological origin, in a continuous or batchwise manner.

Another object of the invention is to provide an electroosmotic process which makes it possible to purify, desalt, and/ or concentrate hog cholera antiserum, and other colloidal or non-colloidal materials, as well as fractions obtained from such materials.

Another object of the invention is to provide an electroosmotic process which makes it possible to selectively concentrate colloidal components of a solution, separating them from lower molecular weight components.

Another object of the invention is to provide apparatus suitable to carry out said electroosmotic process.

Other objects and advantages will be apparent from a consideration of the specification and claims.

Hog cholera antiserum is a standard product of commerce, obtained by bleeding hogs previously immunized with the hog cholera virus. The active principle of this antiserum is believed to be the antibodies of the virus,

`contained in the globulin fraction of the serum. Active fractions can be obtained in a number of Ways well familiar to the art of serum fractionation, and globulin fractions obtainable by the process of the above recited Pat. No. 3,079,313, are to be considered among them. While hog cholera antiserum and its fractions were used in some examples cited, the invention should not be considered as restricted to these products only, as there are many other colloidal or non-colloidal products of biological or other origin to which the invention is equally well applicable.

In accordance with the invention, hog cholera antiserum, its fractions, or other suspensions or solutions are placed within a sample compartment whose two opposing sides are constituted by ion-permeable membranes. Said membranes can be semipermeable membranes of the type of regenerated cellulose, available in commerce under the tradename of Visking Equally satisfactory are other membranes presently available in commerce or in experimental development stages, and manufactured for purice poses of dialysis, ultraltration and related processes, such as cellophane untreated for Water repellency, cyprophane membranes, various membranes containing polyelectrolytes or ion exchange resins, and various membrane filters having controlled porosities and permeabilities, The ion selectivity of the membranes is not critical, and the only requirement of the membranes is that they allow the passage of electrical current, and have pores small enough to retain the desired component of the solution being treated, which, in the case of hog cholera antiserum, is believed to be its globulins.

The optimum size of said pores depends to a certain extent on the material of the membranes and the material treated. Generally, it will be in the range of about 5 to 30, preferably about 10 A.

The electroosmosis sample chamber is exposed to a direct electrical current applied across the membranes by means of two electrodes external to the sample chamber, and electrical contact is established between these electrodes and the membranes by means of an external aqueous medium. More than one sample chamber can be exposed at the same time to one pair of external electrodes, and in such multichamber arrangement of essentially parallel membranes, each sample chamber has to be separated from the next one by a compartment containing the external aqueous medium. Alternatively, separate envelopes of ion-permeable membrane can be immersed in a bath of the external aqueous medium.

The application of the electrical current will generate heat, which can be dissipated by causing circulation of either the sample to be concentrated, or the external aqueous medium, or both, through external heat exchangers. Direct means of cooling the electrodialysis chamber are also possible.

The essential element of the invention consists in including into said external aqueous medium, bathing the membranes of the sample chambers, a polyionic substance detined for-Athis purpose as any molecule or particle containing as part of its chemical structure, and under the pH conditions of its use, a large number of either negatively or positively charged ionized groups, said membranes being so chosen as to have pore sizes sufliciently small to completely retain or substantially retard said polyionic substance. Typical examples of such polyionic substances are polyelectrolytes, such as salts of polyacrylic acid, polysilicic acid, polyphospphoric acid, poly- (ethylene-maleic acid), and poly(methyl vinyl ethermaleic acid). Substances herein referred to as polyionic substances are also described as polyelectrolytes in The Encyclopedia of Polymer Science and Technology, Interscience Publishers, New York, N.Y., vol. 10, p. 781. Polyacrylic acid, polyphosphates and polysilicates are standard products of commerce, while the last two polyelectrolytes are commercially available as anhydrides, and the corresponding salts can be readily prepared by neutralizing their aqueous suspensions with the desired base, for instance sodium or ammonium hydroxide. These polyionic substances exert considerable buffering action over a wide pH range, and their pH can be adjusted to any desired pH within the range of about 5-9, preferably to that corresponding to the sample to be concentrated. The detailed structure of the polyionic substance is unimportant; they do not have to be linear polyelectrolytes, as the five samples cited above, but may be also branched or crosslinked polyelectrolytes of organic or inorganic nature, as employed e.g. in ion exchange resins, either in true solution or in suspension, or they may even be constituted by as simple a molecule as citric acid. The only essential element is the large number of either negatively or positively charged ionized groups.

The polyionic substances are effective over a wide range of concentrations. The maximum concentration that can be used is limited by the great increase in concentration that some polyelectrolytes, especially of the linear type, confer to their suspensions. As water is adsorbed from the sample to be concentrated, the outer aqueous medium containing the polyionic substance becomes progressively more dilute. With this progressive dilution, a gradual loss of effectiveness is observed. The `gross concentration of polyionic substance in the outer aqueous medium is not, necessarily, however, a measure of its effective concentration, as there is retention of the polyionic substance within the apparatus, as described in the following paragraph. Generalizing, the most useful concentration range of the polyionic substances was found to be in the range of 0.07 to 8% by weight.

When such a solution or suspension containing polyionic substances is exposed to the D.C. electrical current in an arrangement as described above, the polyionic substance Will accumulate in a layer of increased concentration in the immediate neighborhood of the ion-permeable membrane interposed in the path of its electrophoretic migration, according to principles partly expounded in my Pat. No. 3,079,318 and the resulting polarization of charged groups against the neutral membranes will convey to it all the characteristics of an ion exchange membrane. When this occurs, a newly discovered empirical observation is that there will be a rapid ilux of Water and ions from the sample compartment into the polyionic substance containing external aqueous medium, through the interposed membrane. The mechanism of this phenomenon is not completely understood, but obviously, the polarization of the polyionic substance in the neighborhood of the membranes causes an electroosmotic gradient to arise across the membranes, which, in turn, causes the inux of water and ions. Moreover, the empirical observation is that the water flux is related to that of the diifusible ions, and if the sample contains no ditfusible ions, such as sodium chloride, then there will be little or no water ux. The polyionic substance may be directly in contact with the electrodes, or an interposing layer of conducting liquid, bounded by ion-permeable membranes, may be present.

In the simplest operational procedure, concentration and desalting will proceed simultaneously, until either near complete dehydration or near complete desalting occurs, at which time the process loses eiectiveness. If dehydration is to be continued past near complete desalting, then periodic or continuous addition of salts to the sample is necessary. Alternatively, if desalting is to be continued past the desired degree of dehydration, then periodic or continuous addition of water to the sample is necessary.

The above described operations can be carried out in three modes, continuously, semicontinuously, or batchwise. In the continuous mode, the sample is circulated only once through the apparatus, While in the semi-continuous mode, the sample is repeatedly circulated through the apparatus, thus achieving a cumulative eiect. In either mode, only a small part of the sample is exposed at any one time to the eiect of electroosmosis, namely, only that part contained in the apparatus. In the batchwise mode, all of the sample is contained in the apparatus, and is exposed to the effect of electroosmosis against the outer polyionic substance containing aqueous medium, the process being terminated when the desired degree of concentration and/ or desaltirrg has been achieved.

Dialysis or electrodialysis is frequently employed to remove a dialyzable impurity from a colloidal solution. Ionic components are only one example of such impurities. lIt is obvious that the described process can be employed in the same manner, allowing dialysis or electrodialysis of any impurity to proceed simultaneously with the above described desalting and dehydration.

An apparatus suitable for carrying out the invention is shown in the accompanying drawings, in which:

FIG. 1 is a detailed vertical sectional view of a cell assembly taken along the lines 1--1 of FIG. 2;

FIG. 2 is a transverse detailed sectional View taken along the lines 2-2 of FIG. 1;

FIG. 3 is an exploded perspective view showing a plurality of the cell structures of FIG. 1, and

FIG. 4 is a vertical sectional view showing a modified form of a suitable apparatus.

Referring first to FIG. 1 of the drawings, this arrangement has a certain similarity to the cell assembly shown in FIG. 4 of my patent 3,079,318. Instead of cells divided by a filter element, the assembly is built up by ion-permeable membranes 14 spaced apart by spacer plates 12. A vessel 1 contains electrolyte solution which preferably is circulated, and electrodes 3 are suitably mounted therein. The electrolyte solution establishes contact between the electrodes and the ion-permeable membranes.

As shown in FIGS. 2 and 3, Windows 16 are formed in said spacers so as to expose the membranes held therebetween at one side to the sample solution to be treated and at the other side to the polyionic substance containing medium so as to produce the desired electroosmotic phenomenon. For this purpose, the spacer plates are provided at the top and bottom with registering ports 18, 20, 22 and 24 which are connected to feed inlets and to outlets for the two solutions, respectively. For the sake of simplicity, only one of said inlets and outlets is shown. Channels 26, 28 connect the ports with trenches 30, 32, from which passages 34, 36 pass the respective liquid to the respective side of the exposed membrane 14. Said membranes have been shown rather thick in relation to the spacer plates. Generally, they are very thin foils.

FIG. 4 illustrates another type of apparatus where a cel 40, open at its top is made from an ion-permeable membrane and receives the aqueous system 42 to be treated. The cell is suspended in a bath of the outer aqueous medium 2, which preferably is circulated across the membrane faces. A plurality of such cell bags can be arranged in a common vessel 1, whereby any suitable kind of arrangement such as side by side or tandem, can be employed. The electrodes 3 are so arranged as to provide an electric field across the whole assembly.

In all cell assemblies, parts of several cells may be arranged in parallel, in series, or in any combinations thereof.

It will be obvious that, for continuous operation, the cell assemblies can be connected with supply systems feeding the aqueous system to be treated, or the aqueous medium containing the electrolyte, or both through the cell assembly. If the liquids are recycled, it is of advantage to provide for cooling by means of external or internal heat exchangers.

The following examples are given to illustrate but not to limit the invention.

EXAMPLE 1 This example is given to illustrate how my electroosmotic process is applied to concentrate a serum and to give a product with 15% final protein concentration, in a semi-continuous manner.

Ten liters of hog cholera serum, protein concentration 5.5%, were submitted to electroosmosis in a three-compartment assembly, each sample compartment having an eective membrane area of 900 cm?, and a volume of about 300 cm. As the external aqueous medium, 50 liters of 3% polyacrylic acid, neutralized to pH 7.5 with sodium hydroxide, were employed. Both, protein and the polyacrylic acid, were recirculated through external exchangers, so as to maintain an inllowing temperature of liquids below 10 C. A D.C. electrical current of a potential of 6 volt/ cm. was maintained throughout the run. Upon overnight electroosmosis, the process was interrupted when the protein concentration in the sample has reached 15%, as determined by measurements of the index of retraction of the sample. Final volume was 3.5 liters.

EXAMPLE 2 This example is` given to illustrate how the electroosmotic process is applied to a diluted serum fraction to give a concentrated final product, with addition of salt, inra batchwise manner.

Fifty ml. of a globulin fraction prepared according to U.S. Pat. No. 3,079,318, from hog cholera antiserum, were exposed to Velectroosmosis in a single compartment chamber, with a membrane area of 100 cm.2. The sample was not recirculated, and only the polyelectrolyte was cooled by recirculating it through an external heat exchanger. The polyelectrolyte solution contained 3% of po1y(ethylenemaleic anhydride), hydrolyzed and brought to pH 7.5 with sodium hydroxide. The applied potential was 8 volt/ cm. The initial current was about 3 amps, but it gradually decreased to below 1 amp. indicating the desalting of the sample. As soon as this amperage was reached, 3 ml. of 15% of sodium chloride solution was added to the sample, resulting in a raise of amperage above the l amp value. This addition was repeated two more times, whenever the current descreased below the 1 amp limit. Near complete dehydration was achieved in 1 hour. The initial protein concentration was 0.5%, and the iinal concentration was 4.7%, the volume of the sample having been reduced to about 4 ml.

EXAMPLE 3 This example is given to illustrate how my electroosmotic process is applied to the concentration of a dilute serum fraction, with the simultaneous purification of the sample through dialytic removal of an impurity, in a semi-continuous manner.

One liter of dog serum, obtained from a dog hyperimmunized against hepatitis and distemper, was precipitated with 3.5 liters of a 0.4% rivanol solution. Rivanol is the trade name for the dye Z-ethoxy-6,9-diaminacridinelactate, commonly used in the art of serum fractionation. The precipitate was discarded, and 4 liters of supernatant were processed against three successive batches of 2% polyacrylic acid, adjusted to pH 7.5 with sodium hydroxide. A total of 30 liters of polyacrylic acid solution was employed. An assembly of iive sample compartments, each with an area of about 100 cm.2, was employed, and both sample and polyelectrolyte recirculated through external heat exchangers. The potential applied was 8 volts/ cm. The concentration proceeded at the rate of about 200 mL/hour, and rivanol was found to electrodialyze rapidly through the membranes, forming yellow complex with the polyelectrolyte and precipitating in the external medium. This is the reason why batches of the polyelectrolyte were employed. After 20 hours, the sample was reduced to 400 ml., and was free of rivanol, as judged by absence of characteristic color. The protein concentration, from an original concentration of 0.24% was increased to 2.3% in the iinal concentrated sample.

EXAMPLE 4 This example is given to illustrate how the invention is applied to a small volume of a diluted enzyme, to give a concentrated final product, without addition of salt, in a batchwise manner.

Five ml. of rabbit muscle lactic acid dehydrogenase containing 3% ammonium sulfate was placed in a single compartment membrane chamber, with a membrane area of 22 cm?. The sample was not circulated, nor was the outer polyelectrolyte cooled. The polyelectrolyte contained 8%, of polyacrylic acid, brought to pH 7.0 with sodium hydroxide. The applied potential was 7 v./cm. Current was 1.2 amps. After 15 minutes of dehydration, 0.5 ml. of liquid was left in the membrane compartment. This was removed and assayed by colorimetry at 520 m1. after color development by the NAD-INT reaction. Whereas the initial sample had given an absorbance value of 0.19 O.D. units, the ten-times concentrate gave an absorbance value of 0.43 O.D., (after 1:3 dilution) indicating recovery.

Similar concentrations have been obtained with the following other materials of biological origin: Rabbit muscle lactic acid dehydrogenase, beef heart lactic acid dehydrogenase, urine, placental extracts, hemoglobin, starch, soybean proteins, E. coli suspensions, celery extracts, botulinum toxin, ribonncleic acid, edestin, beta lactoglobulin, rebonuclease, and others.

EXAMPLE 5 This example is given to illustrate how the invention is applied to a protein solution to give a concentrated iinal product, in a batchwise manner using a simple polyionic molecule as the electro-osmotic medium.

3.6ml. of 5% sodium chloride solution containing 0.5 ml. of rabbit serum was placed in a single compartment membrane chamber of the type shown in FIG. 2 having window area of 18 cm?. The electroosmotic medium was 1.65% sodium citrate. At 4.4 v./cm., starting amperage was 660 ma., rising to 730 ma. as temperature increased over a 30 minute run, at the end of which 1.5 ml. of protein solution remained in the cell.

EXAMPLE 6 This example is given to show that the invention is not limited to colloidal systems but can be applied to solutions and suspensions of any kind. The example demonstrates the increased rate of desalting achievable with the use of polyionic substances in accordance with the invention over either dialysis or electrodialysis in absence of the polyionic substance. It illustrates the use of the process in a continuous manner.

An assembly of five sample compartments, each with an area of about cm?, was employed. Both sample and the external laqueous medium were circulated only once through the apparatus, and the decrease in salt concentration of efliuent sample Versus intiowing sample measured by their electrical conductance. The sample was a solution of ammonium sulfate of concentration indicated in Table I. In the same apparatus, it was iirst exposed to dialysis, then electrodialysis, both of these against tap water, and finally it was exposed to electroosmosis against tap water with 3% polyacrylic acid added, neutralized to pH 7 with sodium hydroxide. Comparative eiciency of the three processes as indicated in Table I.

TABLE I Percent salt removal in single pass through the apparatus Electro- Electroosmosis Dialysis dialysis with polytap Water tap water electrolyte The data clearly show that electroosmosis against polyelectrolyte solution is more effective than against tap water, the effect 'being most marked at lower sample salt concentrations. Ammonium sulfate solutions were used, as this is a common protein precipitating agent.

I claim:

1. A method for electroosmotic concentration, purification, fractionation, and/or desalting of aqueous systems including solutions and suspensions, comprising subjecting said systems to electroosmosis and electrodialysis in contact with one side of a non-ion-selective-ionpermeable membrane, the other side of said membrane being in contact with an outer aqueous medium containing a polyionic substance in solution or suspension, said polyionic substance in said suspension or solution containing as part of its chemical structure, a large number of charged groups, said membrane being so chosen as to have pore sizes sufficiently small to completely retain or substantially retard the transport of said polyionic substance, and the polarity and intensity of the applied Sample, percent concentration direct electrical current being so chosen as to bring about the electrophoretic migration of said polyionic substance towards said dialyzing membrane.

2. A method as claimed in claim 1, wherein said polyionic substance is a polyelectrolyte.

3. A method as claimed in claim 2, wherein said polyelectrolyte is a member of the group consisting of polyacrylic acid, poly(ethylenemaleic acid), poly (methyl vinyl ether-maletic acid), polysilicic acid, polyphosphoric acid, and salt thereof.

4. A method as claimed in claim 2, wherein the concentration of said polyelectrolyte in said outer aqueous medium is in the range of 0.07 to 8 percent by weight.

5. A method as claimed in claim 1, wherein said system contains a colloid of biological origin.

6. A method as claimed in claim 1, wherein said colloid is a member of the group consisting of sera, serums, antiserums, plasma fractions derived therefrom, and globulin fractions.

7. A method as claimed in claim 6, wherein said antiserum is a member of the group consisting of hog cholera antiserum and fractions thereof.

8. A method as claimed in claim 1, wherein said system contains l1/2 to 4 percent by Weight of a salt which is diiusable through said ion-permeable membranes.

'9. A method as claimed in claim 8, wherein the concentration range of said diiusable salt is maintained in said system 'by means of addition of a more concentrated salt solution.

10. A method as claimed in claim 1, wherein said polyionic substance is an organic compound with at least three negatively charged ionized groups in its molecules.

11. A method as claimed in claim 1, wherein said outer aqueous medium has an essentially neutral pH.

12. A method as claimed in claim 1, wherein said outer aqueous medium is adjusted to the pH of said system.

13. A method as claimed in claim 1, wherein said ionpermeable membranes are semipermeable regenerated cellulose membranes.

14. A method as claimed in claim 1, wherein said ionpermeable membranes have polyelectrolytes incorporated in them.

15. A method as claimed in claim 3, wherein said dialyzing membranes have a pore size sufficient to permit easy passage of water and simple ions but to prevent passage of a desired colloid in said system.

References Cited UNITED STATES PATENTS 2,721,171 10/ 1955 Arnold et al. 204-180 P 3,582,488 6/ 1971 Zeineh 204-180 R 1,915,568 6/1933 Gortner et al. 204-180 X 2,547,231 4/ 1951 Sartakoff 204-180 3,051,640 8/ 1962 Traxler 204-180 3,165,415 l/ 1965 Kilburn et al. 99-105 3,394,068 7/ 1968 Calmon et al. 204-180 FOREIGN PATENTS 151,002 10/ 1921 Great Britain 204-180 73 3,234 7/ 1955 Great Britain 204-180 JOHN H. MACK, Primary Examiner A. C. PRESCOTI, Assistant Examiner U.S. Cl. X.R. 204- R, 301 

